Apparatus and method for chemical imaging of a biological sample

ABSTRACT

In one embodiment, the disclosure relates to a method for determining illumination parameters for a stained sample, the method may include providing a stained sample and obtaining an absorption band of the sample; obtaining an emission band of the sample and determining the illumination parameters for the sample as a function of the absorption band and the emission band of the sample.

The instant application is a divisional application of application Ser.No. 11/433,650, filed May 15, 2006 and claims benefit of the prioritydate therefrom, which application is a continuation of application Ser.No. 11/097,161, filed Apr. 4, 2005 (now U.S. Pat. No. 7,045,757), whichis a continuation-in-part of application Ser. No. 11/045,081, filed Jan.31, 2005 (now U.S. Pat. No. 7,060,955) by the inventors named herein,the specifications of which are incorporated by reference herein intheir entirety.

BACKGROUND

Spectroscopic imaging combines digital imaging and molecularspectroscopy techniques, which can include Raman scattering,fluorescence, photoluminescence, ultraviolet, visible and infraredabsorption spectroscopies. When applied to the chemical analysis ofmaterials, spectroscopic imaging is commonly referred to as chemicalimaging. Instruments for performing spectroscopic (i.e., chemical)imaging typically comprise image gathering optics, focal plane arrayimaging detectors and imaging spectrometers.

In general, the sample size determines the choice of image gatheringoptic. For example, a microscope is typically employed for the analysisof sub micron to millimeter spatial dimension samples. For largerobjects, in the range of millimeter to meter dimensions, macro lensoptics are appropriate. For samples located within relativelyinaccessible environments, flexible fiberscopes or rigid borescopes canbe employed. For very large scale objects, such as planetary objects,telescopes are appropriate image gathering optics.

Regardless of the type of optical equipment, a first step in anyspectroscopic investigation is defining a suitable wavelength forilluminating the sample. The step of defining a suitable wavelength forilluminating the sample becomes even more important when simultaneousmultiple images of the sample are sought. Conventional methods suggestilluminating a sample with a first wavelength (e.g., NIR or VIS) toobtain a first image, followed by illuminating the sample with a secondwavelength to obtain a second image (e.g., Raman or dispersive Raman).Consequently, the conventional process is time consuming and is notsuited for simultaneous imaging of the sample. There is a need for anapparatus and method for determining illumination parameters of a samplea priori of illuminating the sample.

The current disclosure addresses the need described above. In oneembodiment, the disclosure relates to a method for obtaining a chemicalimage of a biological sample by providing a biological sample labeledwith a Fluorophore; irradiating the sample with photons havingwavelength within the illumination wavelength range; obtaining aspectral image of the sample; and generating a chemical image from thespectral image. The chemical image may define at least two spectralimages of the sample obtained simultaneously. The spectral images caninclude a Raman image and a fluorescent image.

In another embodiment, an apparatus for obtaining a spectral image of abiological sample comprising means for determining a range ofillumination wavelengths, the illumination wavelength interacting withthe sample to simultaneously provide a first and a second spectra of thesample; a photon source for directing photons with a wavelength withinthe range to the sample, the illuminating photons interacting with thesample to produce interacted photons; a tunable filter for receivinginteracted photons and forming a spectral image of the sample.

In still another embodiment, the disclosure relates to a system forobtaining multiple spectra of a biological sample. The system caninclude a processor programmed with instructions to determineillumination parameters of the sample as a function of the emissionbandwidth of said sample; an illumination source for directing photonshaving a wavelength within the illumination parameters of the sample,the illuminating photons interacting with the sample to provideinteracted photons; and a tunable filter for receiving the interactedphotons from the sample and providing at least a first and a secondspectra of the sample.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 graphically illustrates the relationship between intensity andwavelength of a sample;

FIGS. 2A-2G each schematically illustrate spectral images of a samplereceiving different excitation wavelengths; and

FIG. 3 is a functional diagram of a system according to one embodimentof the disclosure.

DETAILED DESCRIPTION

The disclosure generally relates to a method and apparatus fordetermining illumination parameters for a sample. Having an a prioriknowledge of an optimal illumination parameters (e.g., optimalillumination wavelength range) for obtaining spectral images of a sampleis particularly important in that the optimal illumination parameterenables simultaneous detection of more than one spectra of the sample.The optimal illumination parameters can also be used with differentdetection modes such as: wide field, Raman chemical imaging, multipoint,dispersive single point and dispersive line.

FIG. 1 graphically illustrates the relationship between intensity andwavelength of a sample. The method of obtaining absorption andemissivity bands are conventionally known. It is also known thatemissive energy is associated with fluorescent imaging and absorptionenergy is associated with near infrared (“NIR”) energy. Thus, as a firststep a sample may be illuminated with photons of different frequencies(interchangeably, detection photons or illumination photons) todetermine the sample's absorption and emission wavelengths.

In FIG. 1, line 125 represents the energy absorption relationship of asample exposed to detection photons. Peak 130 indicates the peakwavelength (λ_(Abs, P)) for absorption spectrum of the sample; peak 140indicates the peak wavelength for the emission spectrum (λ_(m)) of thesample and peak 150 shows the peak Raman intensity occurs at (λ_(H, P)).A range of wavelength were absorption energy of the sample can bedetected is shown to extend from λ_(abs-L) to λ_(abs-H). Similarly, arange of wavelength were emissive energy of the sample can be detectextends from λ_(Em-L) to λ_(Em-H).

As will be discussed in greater detail, according to one embodiment ofthe disclosure, an optimal wavelength for multi-spectral imaging canoccur at a wavelength just below or about λ_(abs-L). Thus, a method isdisclosed for defining illumination parameters which includes (i)defining a range of absorption wavelengths for the sample; (ii) defininga range of emission wavelengths for the sample; and (iii) assessing asuitable illumination parameters for the sample as a function of theabsorption wavelength and the emission wavelength. These steps can beimplemented sequentially or simultaneously. By way of example, theregion shown as 155 in FIG. 1 shows a possible illumination wavelengthsuch that it is shorter than the wavelength of a peak in the emissionspectrum. The illumination parameters may also be used to define aillumination laser line or a suitable Raman illumination wavelength.Since wavelength and frequency are inversely proportional, steps(i)-(iii) can be implemented and defined in view of a frequency band.That is, in view of a range of absorption wavelengths of a sample anequivalent frequency bandwidth for the sample can be defined.

In another embodiment of the disclosure, a method for determiningillumination parameters for a sample includes: simultaneouslyilluminating the sample with illuminating photons. The illuminatingphotons can have several different wavelengths or define a broad rangeof wavelengths. Next, the emissive and absorption wavelengths for thesample can be defined. Alternatively, the sample's bandwidth foremission and absorption can be determined. The emission and theabsorption bands can also define the peak intensity wavelength as wellas the lower and the upper wavelength ranges for each band. Using thelower wavelength of the absorption band (λ_(abs-L)) as a starting point,an optimal Raman wavelength detection wavelength for the sample can bedefined as Raman scattered photons having wavelength about or belowλ_(abs-L). By way of example, one such region is shown as region 155 inFIG. 1. The illumination parameters thus obtained can be used toilluminate the sample with illuminating photons of different wavelengthsto obtain simultaneous spectral images of the sample. The illuminatingphotons can be provided by a laser line, wide-field, Raman chemicalimaging, multipoint imaging, dispersive single point and dispersivelines specifically devised to be within the desired wavelength range.

FIGS. 2A-2G each schematically illustrate spectral images of a samplereceiving different excitation wavelengths. More specifically, FIGS.2A-2G depict absorption, emission and Raman spectra for a biologicalsample stained with a die and a method for determining illuminationparameters for the sample in view of absorption and emission spectra ofthe sample. In one embodiment of the disclosure the die is a Flourophor.Suitable Fluorophore stains include an immuno-fluorescent compound, abasophilic compound, an acidophilic compound, neutral stains andnaturally occurring luminescent molecules. Once stained, the sample canbe irradiated with photons having a wavelength within the illuminationwavelength range in order to obtain the spectral images of the sample.

In FIG. 2A, peak 100 shows the emission peak for the stained sample. Asis conventionally known, the emission bandwidth (or its equivalent rangeof wavelength) is a property of the material. In FIG. 2A, the emissionrange spans between λ_(A)-λ_(B), with a peak emission wavelengthoccurring at λ_(P). The illumination (excitation) wavelength isarbitrarily set at λ_(X1). Raman peaks are identified as peaks 160.Raman peaks are shifted from excitation wavelength (λ_(X1)) by a fixedwavelength which is commensurate with the energy lost due to Ramanvibration. Increasing or decreasing the excitation wavelength will havea direct effect on the wavelength where the Raman peaks occur. This isschematically illustrated in FIGS. 2A-2G where changing the excitationenergy from the wavelength λ_(X1) to λ_(X7) results in shifting thewavelength where Raman peaks 160 occur. Referring again to FIG. 2A, theRaman peaks 160 occur at wavelength λ_(X1)−1/ψ; where ψ is the Ramanenergy loss due to Raman excitation expressed in wavenumbers and can bequantified as

ψ=(½Πc)(k/μ)^(1/2); where k is the chemical bond force constant, c isthe speed of light, and μ is the reduced mass of the molecularoscillator.

In FIGS. 2B and 2C, peak 110 represents the emission peak and peaks 160represent Raman scattering peaks for the sample under study. Peak 130illustrates the sample's Fluorescence spectrum. In FIGS. 2B and 2C theexcitation wavelength is set to λ_(X2) and λ_(X3), respectively, suchthat the Fluorescent spectrum of the sample occurs at a wavelength nearthe excitation region as shown. As can be seen from FIGS. 2B and 2C, thesample's Fluorescence spectrum overlaps with the Raman peaks 160, whichas stated, occurs at a fixed wavelength from the excitation wavelength.The overlap makes spectral analysis difficult, if not impossible, as theRaman signals will become overwhelmed by the Fluorescent signals.

In contrast, the illumination parameter λ_(X4) in FIG. 2D is selectedsuch that Raman peaks 160 occur just below the onset of Fluorescentspectrum 130. Here, each of the Raman 160, Fluorescence 130 and Emission110 spectra are visible within a narrow range of wavelengths and can bedetected substantially simultaneously with a single detection device.

In FIG. 2E, the sample has dual Fluorescence peaks 130 and 135.Fluorescence peak 135 defines a lower intensity peak as compared withFluorescence peak 130. Signals from the Raman peaks 160 may be moreeasily detectable over Fluorescent signal indicating peak 135. In FIG.2E, illumination wavelength λ_(X5) is selected such that Raman peaks 160overlap with the Fluorescence peak 135. However, since the Raman signalshave a higher intensity, Raman peaks 160 may be identified from emissionspectrum 135.

In FIG. 2F the excitation wavelength is shifted to λ_(X6) and RamanPeaks 160 are no longer eclipsed by Fluorescent peaks 130 and 135. TheRaman signals may nonetheless receive interference from emissionspectrum of peak 110. Since at least a portion of Raman peaks 160 fallsbetween Fluorescent peak 135 and emission peak 110, the Raman signal maybe at least partially distinguishable. In FIG. 2G, excitation wavelengthλ_(X7) is shifted to a lower wavelength resulting in the shifting ofRaman peaks 160. Here, emission peaks 110 overlap Raman peaks 160rendering the Raman signals indistinguishable from the emission signals.

As can be seen from FIGS. 2A-2G, the excitation wavelength can beselected such that at least two spectra of the sample are simultaneouslyvisible within a narrow range of wavelengths. According to oneembodiment of the disclosure the excitation wavelength is selected suchthat Raman peaks appear at a wavelength substantially free frominterference from other signals. According to another embodiment, theexcitation wavelength can be selected such that signals from Raman peaksare distinguishable from signals depicting emission or Fluorescencespectra. According to still another embodiment of the disclosure, theexcitation wavelength is selected such that an imaging device cansimultaneously capture the Fluorescence emission as well as the Ramanspectra for the sample. In still another embodiment of the disclosure,the excitation wavelength is selected such that an imaging device cansimultaneously capture the Fluorescence emission as well as the Ramanspectra for the sample.

In one embodiment of the disclosure, an apparatus is provided to assessthe illumination parameter for a histologically labeled sample. Thesample can be labeled with a conventional identifier, such as aFluorophore substance. Next, an illumination parameter defining asuitable illumination wavelength can be selected such that both theemission peak and Raman scattering peaks can be detected with oneimaging apparatus. The imaging apparatus may include gathering optics(e.g., optical trains for collecting photons emitted, Raman scattered,transmitted, or reflected from the sample), one or more tunable filters(e.g., liquid crystal tunable filter (LCTF), Acousto-optical TunableFilter (AOTF) or fiber array spectral translator). A charged-coupleddevice or other suitable camera or recording medium may be coupled tothe imaging apparatus in order to capture the spectra.

In a system according to an embodiment of the disclosure, theillumination parameter for a sample includes one or more illuminationsources, an optical train and a processor programmed with instructionsto simultaneously illuminate the sample with illuminating photons anddetect an emission band and an absorption band of the sample. Theinstructions can also include defining a lower wavelength range and anupper wavelength range for the band and determine the illuminationparameters for the sample as a function of the absorption and theemission bands of the sample. Finally, the instructions may includedefining a suitable Raman wavelength for the sample at a wavelengthshorter than the lower wavelength range (λ_(EM, L)) of the emissionspectrum.

FIG. 3 is a functional diagram of a system according to one embodimentof the disclosure. In FIG. 3, system 300 is devised to obtain andanalyze chemical images of sample 310. Sample 310 can be any biological,organic or inorganic sample suitable for histological studies.Illumination source 330 is positioned to provide excitation photons 332to sample 310. Illumination source 330 can be positioned above, near orbelow the sample. Excitation photons 332 define excitation wavelengthswhich can cover a broad range of spectra (e.g., λ_(X1)-λ_(X7)).Moreover, illumination source 330 can be adapted to change excitationwavelengths based on instructions communicated by Processor 320.Detector 340 is positioned near sample 340 to receive interacted photons342. Interacted photons 342 may include Fluorescence, reflection,absorption, transmission, emission and Raman photons. Interacted photonscan be received and collected by Detector 340 which may includeelectro-optical devices suitable for gathering and analyzing photons ofbroad wavelengths. Detector 340 may include, for example, an opticaltrain for gathering and focusing interacted photons; one or more opticalfilters for rejecting photons of undesired wavelengths; an LCTF forobtaining spectral images of Sample 310; and a charge-coupled device fordevising a chemical image based on the spectral images of Sample 310.Detector 340 can communicate with peripheral network devices 360 such asprinters, video recorders or internet communication systems.

Processor 320 can receive spectral images of the sample from Detector340 and determine whether the illumination wavelength should be changed.For example, if Detector 340 devises a spectral image of a samplesimilar to FIG. 2C, processor 320 can determine that Raman signals frompeaks 160 are indistinguishable from Fluorescent signal 130. Based onthis determination, processor 320 can direct a change in excitationwavelength produced by illumination source 330 such that signalinterference is reduced to that shown in FIG. 2D. Processor 350 can alsocommunicate with CPU 350 to receive executable instructions or to accessa database of related information such as the chemical identifier or theproperties of the Fluorophore used on sample 310. CPU 350 can be used tocommunicate additional information to the processor.

After one or more iterations, Processor 320 can determine the optimalillumination parameter for the sample such that more than one spectralimage can be detected simultaneously. The spectral image of the samplecan be used to define one or more chemical images of the sample and toidentify the sample under study. Such analysis can be implemented usinga single detection and identification system and lends substantialefficiency to histological analysis.

While the principles of the disclosure have been disclosed in relationto specific exemplary embodiments, it is noted that the principles ofthe invention are not limited thereto and include all modification andvariation to the specific embodiments disclosed herein.

1. A method for simultaneously obtaining spectral images comprising:providing a histologically labeled biological sample; identifying arange of illumination parameters as a function of absorption andemission spectra of the labeled sample by irradiating the sample withphotons having wavelength within an illumination wavelength range;selecting illumination parameters within the identified range;illuminating the labeled sample according to the selected illuminationparameters; and simultaneously obtaining at least two spectral images ofthe sample.
 2. The method of claim 1, wherein the step of selectingillumination parameters comprises selecting the optimal illuminationwavelength range providing simultaneous spectral images of the sample.3. The method of claim 1, wherein the step of identifying a range ofillumination parameters further comprises defining a fluorescenceemission peak for the labeled sample and defining the upper limit of therange to be the wavelength corresponding to the emission peak.
 4. Themethod of claim 1, wherein the spectral image of the sample includes atleast one of a fluorescent signal and a Raman signal.
 5. The method ofclaim 1, wherein one of the at least two spectral images of the sampleis selected from the group consisting of fluorescence, reflection,absorption, transmission, optical, and Raman images.
 6. The method ofclaim 1, wherein the sample is histologically labeled with a fluorophoreselected from the group consisting of an immuno-fluorescent compound, abasophilic compound, an acidophilic compound, a neutral stain, and anynaturally occurring luminescent molecules.
 7. The method of claim 6,wherein the acidophilic compound is Eosin.
 8. The method of claim 6,wherein the basophilic compound is Hematoxylin.
 9. The method of claim1, wherein the illumination wavelength range overlaps with a region ofan emission bandwidth. 10-31. (canceled)
 32. The method of claim 1,further comprising the step of generating a chemical image identifyingthe composition of the sample from the spectral images.
 33. A method ofobtaining a Raman image simultaneously with another spectral image, themethod comprising: irradiating a labeled sample with photons to identifythe absorption and emission spectra of the labeled sample; as a functionof the absorption and emission spectra, determining a range ofexcitation wavelengths for providing photons to obtain the Raman imagedistinguishable from the other spectral image; irradiating the labeledsample at an excitation wavelength selected from within the range; andobtaining the Raman image and the other spectral image simultaneously.34. The method of claim 33, further comprising varying the selectedexcitation wavelength until an optimal Raman image is obtainedsimultaneously with the other spectral image.
 35. The method of claim33, further comprising generating a chemical image identifying thecomposition of the sample from the Raman image and the other spectralimage.
 36. The method of claim 33, wherein a lower limit of theexcitation wavelength range is defined to be near the lowest wavelengthcorresponding to the absorption spectra of the labeled sample.
 37. Themethod of claim 33, wherein an upper limit of the excitation wavelengthrange is defined to be the wavelength corresponding to an emission peakfor the labeled sample.
 38. The method of claim 33, wherein the otherspectral image of the labeled sample is selected from the groupconsisting of fluorescence, reflection, absorption, transmission,optical, and Raman images.
 39. The method of claim 33, wherein thesample is labeled with a fluorophore.
 40. The method of claim 39,wherein the fluorophore is selected from the group consisting of animmuno-fluorescent compound, a basophilic compound, an acidophiliccompound, a neutral stain, and any naturally occurring luminescentmolecules.
 41. The method of claim 40, wherein the acidophilic compoundis Eosin.
 42. The method of claim 40, wherein the basophilic compound isHematoxylin.
 43. The method of claim 33, wherein the selected excitationwavelength overlaps a region of an emission bandwidth.
 44. The method ofclaim 33, wherein the selected excitation wavelength overlaps a regionof an absorption bandwidth.